The long term goals of this proposal are to determine the molecular mechanisms of amyloid formation in Alzheimer's disease and provide the molecular basis for the design of drugs that could prevent amyloid formation. Amyloid beta protein (Ab) is the principal component of poorly soluble extracellular amyloid depositions found in the brains and cerebrovasculature of patients with Alzheimer disease, Down Syndrome, hereditary cerebral hemorrhage and in normal aging. However, self-aggregating and cytotoxic Ab appears to be soluble and non-toxic in cerebrospinal fluid (CSF) and serum of patients and healthy individuals. To explain this apparent contradiction, we hypothesized that, normally released into extracellular space, Ab is sequestered by extracellular proteins which prevent self-aggregation, amyloid formation and cytotoxicity of Ab. Recently, we found that two Ab binding proteins, transthyretin (TTR) and apolipoprotein E (apoE), inhibit Ab amyloid formation in vitro. The current proposal is based on this observation and is focused on the elucidation of molecular mechanisms of interactions of TTR and apoE with Ab. The achieved goals will provide a basis for rational design of compounds that could prevent amyloid formation and Ab cytotoxicity in vivo. Our specific aims are: 1. To determine contact sites of TTR that are involved in the inhibition of Ab amyloid formation in vitro. An integrated approach will employ structural biological data, computer assisted modeling and biochemical analysis of amyloid formation by recombinant TTR mutants. Coexpression of recombinant TTR mutants and Ab in transgenic C. elegans will be used to verify these putative sites in vivo. 2. To determine contact sites of apoE that are involved in inhibition of Ab amyloid formation in vitro. An integrated approach will employ structural biological data, computer assisted modeling and biochemical analysis of inhibition of amyloid formation by recombinant apoE mutants. coexpression of recombinant apoE mutants and Ab in trangenic C. elegans will be used to verify these putative sites in vivo. 3. To determine the molecular mechanism of inhibition of Ab amyloid formation using TTR and apoE and synthetic peptides that correspond to the identified contact areas. 4. To determine the effect of TTR and apoE and synthetic peptides that correspond to the identified contact areas on cytotoxicity of Ab using cultures of rat PC12 pheochromocytoma cells. Elucidation of molecular interactions of TTR and apoE with Ab will provide a basis for the design of drugs that could prevent amyloid formation and block Ab cytotoxicity.